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Virus Research 153 (2010) 143–150

Contents lists available at ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

ccult hepatitis B virus infection with low viremia induces DNA damage,poptosis and oxidative stress in peripheral blood lymphocytes

rpit Bhargavaa,b, Saba Khana, Hariom Panwara, Neelam Pathaka, Ram P. Pundea,ubodh Varshneya, Pradyumna K. Mishraa,b,∗

Bhopal Memorial Hospital & Research Centre, Bhopal, IndiaDivision of Translational Research, Tata Memorial Centre, ACTREC, Navi Mumbai, India

r t i c l e i n f o

rticle history:eceived 17 May 2010eceived in revised form 17 July 2010ccepted 19 July 2010vailable online 4 August 2010

eywords:ccult infectionsepatitisNA damage responseirculating nucleosomes

a b s t r a c t

Occult HBV infections (OHBI) are often associated with poor therapeutic response and increased riskof developing hepatocellular carcinoma. Despite a decade of research, OHBI still remains an intricateissue and much is yet to be defined about their possible immune implications. As HBV is known to infectperipheral blood lymphocytes, the present study aimed to explore the molecular mechanisms underlyingDNA damage response triggered due to OHBI in host cells. The study was divided into three groups i.e.group A (OHBI patients n = 30, viral load ≤100 IU/mL); group B (chronic HBV patients, n = 30) and groupC (controls, n = 30). Peripheral blood lymphocytes were isolated and DNA damage response, apoptosisand oxidative stress were the studied parameters. A significant increase in the phosphorylation of DNAdamage response proteins (ATM, ATR, H2AX and p53) in OHBI in comparison to controls suggested thatOHBI induces DNA damage in peripheral blood lymphocytes and elicit a PI3 kinase mediated cellular

xidative stress response. In addition, increased DNA fragmentation, circulating nucleosome levels and mitochondrialmembrane depolarization observed in OHBI group indicated that this damage might lead to cellulardemise and immune hypo-responsiveness. Moreover, OHBI was also observed to be strongly associatedwith oxidative stress as suggested by the augmented levels of DCF fluorescence and depleted GR activity.Collectively, these results provide the basic knowledge about the genotoxic effects of OHBI in peripheralblood lymphocytes. Such studies may possibly open up new avenues for identifying novel therapeutictargets for viral hepatitis.

. Introduction

HBV is considered as one of the most prevalent viral pathogensn humans and main etiological agent for chronic hepatitis, cirrhosis

nd HCC. It is estimated that worldwide around 2 billion peoplere infected with HBV among which 350 million are chronicallynfected (World Health Organization, 2008). Approximately, 40%f these patients develop cirrhosis, liver failure, or HCC and over 1

Abbreviations: anti-HBc, antibodies to hepatitis B core antigen; DAPI, 4′ ,6-iamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminete-raacetic acid; ELISA, enzyme-linked immunosorbent assay; FITC, fluoresceinsothiocyanate; HBV, hepatitis B virus; HbsAg, hepatitis B surface antigen; HCC,epatocellular carcinoma; IFN, interferon; IL, interleukin; PBS, phosphate bufferaline; PI3 kinase, phosphoinositide 3 kinase; SDS-PAGE, sodium dodecyl-sulfateolyacrylamide gel electrophoresis; TNF, tumor necrosis factor.∗ Corresponding author. Present address: Division of Translational Research,dvanced Centre for Treatment, Research & Education in Cancer, Tata Memorialentre, Kharghar, Navi Mumbai 410 210, India. Tel.: +91 022 27405121.

E-mail address: pkm 8bh@yahoo.co.uk (P.K. Mishra).

168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2010.07.023

© 2010 Elsevier B.V. All rights reserved.

million die annually of HBV-related chronic liver diseases (Wright,2006).

Progression of HBV infection is considered as an activephenomenon with inconsistent biochemical, virological and his-tological outline at different stages with an unpredictable pace(Bertoletti and Gehring, 2006; Lai and Yuen, 2007). It is believed thatapart from different biological variants, pathogenesis and clinicaloutcome of the infection is widely influenced by host immunologi-cal factors (Liu, 2001; Baumert et al., 2007) and any perturbations inthem can lead to viral persistence (Khettry et al., 2000; Michalak etal., 2000). Despite a decade of research, HBV infection is consideredas a multifaceted disease and a lot remains to be defined about cer-tain of its more complex forms such as occult infections. Occult HBVinfections (OHBI) are those infections which lack detectable serumHBsAg, but are positive for the presence of HBV DNA (Raimondo

et al., 2008). The main mechanism through which occult infectionoccurs is still not completely understood, however, several possibleexplanations, such as integration into human genome and mainte-nance in peripheral mononuclear cells (PBMC), exist (Hou et al.,2001; Hu, 2002; Tamori et al., 2003). The attachment site of the

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44 A. Bhargava et al. / Virus

irus for PBMC has been identified in the preS1 encoded proteinf the virus envelope, the same involved in hepatocytes infectionPontisso et al., 2008). Although, active replication of HBV in thesextra hepatic sites is controversial, some studies in acute infectionsave shown the presence of covalently closed circular DNA andssential viral poly-adenylated mRNAs, which is a precondition foriral replication (Stoll-Becker et al., 1997; Murakami et al., 2004).

Earlier studies on various other viruses have shown that viraleplication exposes host cells to a large amount of exogenousenetic material, which acts as stress factor to elicit DNA damageignaling cascade finally leading to apoptosis (Sinclair et al., 2006;iu et al., 2008). In addition, a study has also demonstrated thathese signaling mediators are manipulated by HBV for its repli-ation and propagation (Zhao et al., 2008a). Although, such DNAamage cascade is exhaustively studied in hepatic cells and someeports have also shown that chronic HBV infection leads to DNAamage in lymphocytes (Martin-Lluesma et al., 2008; Wang et al.,008), molecular mechanisms underlying genotoxic effects of OHBI

n peripheral blood lymphocytes remain to be elucidated.The aim of our present investigation was to explore the molec-

lar mechanisms underlying the genotoxic effects of OHBI ineripheral blood lymphocytes. In this regard, the OHBI patients

.e. seronegative for the presence of HBsAg but positive for HBVNA (≤100 IU/mL) were undertaken for the study. Separation oferipheral blood lymphocytes was done using density gradiententrifugation and DNA damage response, apoptosis and oxidativetress were the studied parameters.

. Materials and methods

.1. Subject selection

The study was approved by the Institutional Review Board,hopal Memorial Hospital and Research Centre, Bhopal (India).

nformed consent was obtained from all the human subjectsncluded in the study. The study was divided into three groups i.e.,roup A comprised of patients with OHBI (n = 30), group B includedhronic HBV patients (n = 30), group C comprised of age and gen-er matched controls (n = 30). OHBI were defined as those whichere negative for HBsAg, but positive for HBV DNA (≤100 IU/mL)

n serum and peripheral lymphocytes. The diagnosis of chronicBV infections was based on the sero-positivity of HBsAg for last 6onths and was non-cirrhotic with fibrosis stages 2 and 3. Controls

ncorporated in the study were without any clinical history of hep-titis and were considered healthy after routine laboratory analysis.ther possible causes of cellular injury, such as co-infection, alco-ol and drugs were also excluded. 10 mL of EDTA conjugated bloodample was collected from each subject by routine venipunctureethod and was used for viral DNA quantification.

.2. Reagents

Qualitative screening for HBsAg and anti-HBc, was done bysing ELISA kits from Diasorin S.p.A., Saluggia, Vercelli, Italy.uantitative estimation of HBV DNA in blood and peripheral

ymphocytes was performed through Light Cycler 2.0 (Rocheiagnostics, Mannheim, Germany) by using RealArt HBV LC PCRit (Artus GmbH, Hamburg, Germany). Isolation of lymphocytesrom peripheral blood was performed using Lymphosep (MPiomedicals, Solon, OH, USA). Phytohemagglutinin and RPMI

rowth medium were procured from Gibco-BRL Invitrogen Co.Carlsbad, CA, USA). Assessment of DNA damage response wasone by using specific antibodies against Ataxia telangiectasiautated phosphoserine-1981 (p-ATM), Ataxia telangiectasia and

ad3 related phosphoserine-428 (p-ATR), H2AX phosphoserine-

rch 153 (2010) 143–150

139 (�H2AX) and p53 phosphoserine-15 (p-p53) from Abcam,Cambridge, UK and Calbiochem, Nottingham, UK with appropri-ate dilution in 1X phosphate buffered saline (PBS) prior to use.Quantitative measurement of �H2AX was done using H2AX Phos-phorylation Assay kit (Upstate Cell Signaling Solutions, Temecula,CA, USA). To quantify apoptosis, Apoptotic DNA Ladder kit andCell death detection ELISA (CDDE) kit from Roche Applied Sci-ence, Mannheim, Germany was used. Detection of mitochondrialmembrane depolarization was done by using Mitochondrial Mem-brane Potential Detection kit from BDTM Biosciences (San Diego, CA,USA). Determination of ROS was performed by CM-H2DCFDA fromMolecular Probes, Invitrogen Co. (Carlsbad, CA, USA). Depletion ofGlutathione reductase (GR) was evaluated by using ELISA kit fromTrevigen Inc. (Gaithersburg, MD, USA).

2.3. Lymphocyte isolation and culture

Lymphocytes were isolated through density gradient cen-trifugation using Lymphosep and were examined for viabilityusing trypan blue dye exclusion test and counted by Neubaer’shemocytometer. 1 × 106 cells were cultured with 2 mL RPMI 1640media (pH 7.4) supplemented with 10-mM/L l-glutamine, 24 mM/LNaHCO3, 10-mM/L Hepes, 10,000-U/mL penicillin, and 10,000-�g/mL streptomycin. Cultured lymphocytes were mitogenicallystimulated with the addition of 0.2 mL of phytohemagglutinin,followed by incubation at 37 ◦C in 5% CO2 atmosphere with 95%relative humidity for 24 h.

2.4. Quantification of the viral nucleic acid

Extraction of viral nucleic acid (HBV DNA) was done usingDNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according tothe standard kit protocol. Briefly, DNA was isolated by lysis underhighly denaturing conditions followed by binding of viral DNA toa silicagel-based membrane (QIAamp membrane) in the QIAampMini spin column and washing with buffer AW1 and AW2. DNAwas then eluted using DNase-free elution buffer and was detectedusing RealArt HBV LC PCR kit through Light Cycler 2.0 following allthe necessary instructions from the supplier.

2.5. Analysis of DNA damage response

Analysis of �H2AX formation as a marker for DNA damage wasdone through immunofluorescence. For qualitative analysis, cellswere fixed in 10% formaldehyde for 1 h and permeabilized with0.1% Triton X-100 for 30 min, blocked with 3% BSA for 3 h, and thenincubated with primary antibody against �H2AX (dilution 1:1000)for 3 h and FITC conjugated secondary antibody (dilution 1:200)for 1 h, while nucleus was counterstained with DAPI. Preparationswere immediately mounted with anti-fade solution, covered withcover slips and stored in the dark at 4 ◦C. Images were acquiredby a spectral bio-imaging system and analyzed with version4.0 software (Applied Spectral Imaging, Edingen, Neckarhausen,Germany). Quantitative measurement was done using H2AX Phos-phorylation Assay kit and by following the standard kit protocol.Cells were quantitatively assessed by flow cytometer (FACS Cal-ibur, BD-Biosciences, San Jose, CA, USA), fluorescence in the FL1channel being recorded for 10,000 events (Mishra et al., 2008).

The activation of proteins in response to damage was analyzedthrough western blot. Briefly, cells were rinsed twice in PBS andlysed in the buffer (10% SDS, 1 M Tris pH 7.6, 5 mM EDTA). The

obtained cell lysates were centrifuged at 12,000 rpm for 10 minat 4 ◦C and the supernatant was collected. An amount of 100 �gprotein was analyzed through 10% SDS-PAGE and transferred ontonitrocellulose membrane in 25 mM Tris, 194 mM glycine and 20%methanol at 4 ◦C with semi-dryer transfer unit of Hoefer (Hollis-

A. Bhargava et al. / Virus Research 153 (2010) 143–150 145

Table 1Comparative clinical background of occult and chronic HBV patients with controls.

Subjects Age (years) ALT (U/L) AST (U/L) Viral load (IU/mL) Platelet counts (lakhs/mm3) Bilurubin (g/dL)

Control 37 ± 3.81 36.28 ± 2.64 27.5 ± 2.34 – 3.1 ± 0.86 0.82 ± 0.24

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Occult HBV 38 ± 2.54 36.91 ± 2.43 28.2 ± 3.12Chronic HBV 42 ± 3.92 80.1 ± 7.68 58.7 ± 3.98

ata are expressed as mean ± SE and the statistical analysis was done using Studen

on, MA, USA). Membranes were blocked with 5% non-fat milk in.1% PBST and incubated overnight at 4 ◦C with primary mono-lonal antibody (1:1000 dilution) specific for p-ATM, p-ATR and-p53. Membranes were washed two-to-three times for 20 minith 0.1% PBST and incubated for 2 h at room temperature with

lkaline phosphatase conjugated secondary polyclonal antibody1:2500 dilution). Membranes were washed and bound antibodiesere analyzed using ECL Plus (GE Healthcare Pittsburgh, PA, USA).uantitation of the signals was done by using Quanti One software

Bio-Rad, Philadelphia, PA, USA) (Mishra et al., 2009).

.6. Detection of apoptosis

.6.1. DNA ladder profilingTo determine inter-nucleosomal DNA fragmentation (ladder),

ells were treated with 200 �L of lysis buffer for 10 min at 15–25 ◦Collowed by addition of 100 �L Isopropanol. Samples were trans-erred to spin column tubes, centrifuged at 8000 rpm for 1 min,ashed twice and DNA was eluted in pre-warmed elution buffer by

entrifugation at 8000 rpm for 1 min. The samples were dissolved in5 �L of loading buffer and subjected to electrophoretic separationn 1% agarose gel (Mishra et al., 2010).

.6.2. Cell death detection ELISAMeasurement of circulating nucleosomes as the markers of

poptotic cell death was done through CDDE by following all man-facturers’ instructions. The assay is based on the quantitativeandwich-enzyme-immunoassay principle using monoclonal anti-odies directed against DNA and histones. The absorbance kineticsas measured at 405 nm by ELISA reader (Mishra et al., 2010).

.6.3. Mitochondrial membrane potentialMitochondrial membrane potential of the cells was detected

y staining cells with JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-etraethylbenzimidazolcarbocyanine iodide). The assay wasarried out as per supplier’s instructions. The gate was applied inhe FSC/SSC dot plot to restrict the analysis to lymphocytes only.or the gated cells, the ratio of FL1/FL2 was evaluated. In each case,total of 10,000 events were recorded in HI mode (Mishra et al.,008).

.7. Evaluation of oxidative stress

.7.1. Levels of intracellular ROSA fresh stock solution of CM-H2DCFDA (5 mM) was prepared in

MSO and diluted to a final concentration of 1 �M in 1× PBS. Theells were washed with 1× PBS followed by incubation with 50 �Lf working solution of fluorochrome marker CM-H2DCFDA (finalorking concentration adjusted to 2.5 �g/50 �L) for 2 h. The cellsere harvested, washed in PBS, and cell-associated fluorescenceas measured through flow cytometry in FL1 channel (Mishra et

l., 2010).

.7.2. Estimation of GR activityLevels of antioxidant defense system enzyme, GR, were mea-

ured using instructions as supplied by the manufacturer and the

38.1 ± 2.085 2.98 ± 0.41 0.95 ± 0.16676.8 ± 30.88 1.58 ± 0.26 2.14 ± 0.37

st; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

absorbance kinetics was measured at 340 nm through an ELISAreader (Mishra et al., 2010).

2.8. Statistical analysis

The data were analyzed statistically by using Student’s t-test andp ≤ 0.05 was considered to be significant.

3. Results

3.1. Clinical characteristics of patients

OHBI patients were reported HBsAg negative but found to beHBV DNA positive in plasma and peripheral lymphocytes. Thesepatients were also observed to be positive for anti-HBc and theirviral load ranged from 20 IU/mL to 80 IU/mL. The clinical symp-toms of the OHBI patients were namely, abdominal pain, weightloss, fever, nausea, vomiting and diarrhea. The chronic patientswere positive for HBsAg, anti-HBc and HBV DNA and their viralload ranged from 500 IU/mL to 1000 IU/mL. Comparative clinicalbackground of occult and chronic HBV patients with controls hasbeen shown in Table 1.

3.2. DNA damage response

One of the first proteins to be phosphorylated upon DNA damageis the histone variant H2AX, which acts as a signal for recruit-ment of DNA damage proteins to damage point (Kuo and Yang,2008). Immunofluorescence analysis of peripheral blood lympho-cytes from OHBI patients showed an increased phosphorylation ofH2AX at Ser139 (�H2AX) in a punctate nuclear pattern as comparedwith age and gender matched control. Quantitative observationsrevealed that �H2AX formation further increased in chronic infec-tions (Fig. 1A and B). The observation suggested that HBV despiteof its occult state and low viral load (≤100 IU/mL) induces genotox-icity on the immune cells.

The results of western blot analysis revealed that infection withoccult HBV induces DNA damage and elicits PI3 kinase mediatedresponse as suggested by the increased ATR/ATM phosphorylation.A significant increase in the phosphorylation levels of its down-stream substrate p53 at Ser-15 was also reported. These levelsincreased in chronic infection showing a direct effect of diseaseseverity with genotoxicity of HBV on lymphocytes (Fig. 1C). Soft-ware band analysis of DNA damage response proteins showed asignificant increase in their percent expression from 18.4% (p-ATR),14.2% (p-ATM), and 15.1% (p-p53) in controls to 29.9% (p-ATR),35.9% (p-ATM), 46.5% (p-p53) in occult and 38.4% (p-ATR), 45.4%(p-ATM), 56.9% (p-p53) in chronic. Taken together, these resultsindicate that OHBI with low viral load (≤100 IU/mL) leads to DNAdamage and elicit activation of PI3 kinase mediated response path-way.

3.3. Apoptotic index

3.3.1. Increased DNA fragmentationThe typical DNA ladder, a hallmark of apoptosis detected by

agarose gel electrophoresis, demonstrated that occult HBV induces

146 A. Bhargava et al. / Virus Research 153 (2010) 143–150

Fig. 1. (A) Flow cytometric analysis of H2AX phosphorylation showed a significant peak shift in peripheral blood lymphocytes of occult HBV infected patients as comparedwith controls. (I) Control; (II) occult HBV; (III) chronic HBV. (B) Microphotographs (200×) showing immunofluorescence analysis of H2AX phosphorylation with significantp atientr eins (pl of theo

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unctate nuclear patterns in peripheral blood lymphocytes of occult HBV infected pepresents the �H2AX foci. (C) Western blot analysis of DNA damage response protymphocytes of control (I), occult HBV (II) and chronic HBV (III). (For interpretationf the article.)

poptosis in the peripheral blood lymphocytes. A significantncrease in the DNA fragmentation in the peripheral lymphocytesf occult HBV patients was observed as compared with age andender matched controls which further increased in chronic HBVatients (Fig. 2A).

.3.2. Raised circulating nucleosomes levelIn comparison to the controls the observed increase in the lev-

ls of circulating nucleosomes (markers of apoptotic cell death) inHBI patients further strengthen our above observations that these

nfections lead to cellular demise. The mean circulating nucleo-omes levels in controls was 0.096 AU (arbitrary unit) while in OHBInd chronic HBV patients the levels were 0.359 AU and 0.504 AU,espectively.

.3.3. Enhanced mitochondrial depolarizationMembrane-permeable lipophilic cationic fluorochrome JC-1

as used as a probe of transmembrane potential (� ). JC-1 pen-trates into cells and its fluorescence reflects � . As comparedith controls a significant increase in the cells with depolar-

zed mitochondria (loss of � ) was observed in occult HBV.he mean percentage of cells with depolarized mitochondria

s. The nucleus has been counterstained with DAPI (blue) while FITC signals (green)-ATR, p-ATM and p-p53) along with loading control beta-actin in peripheral bloodreferences to color in this figure legend, the reader is referred to the web version

in occult HBV patients were 48.18 ± 4.65%, while in controlsand chronic HBV the levels were 0.81 ± 0.12% and 78.34 ± 8.51%,respectively (Fig. 2B).

3.4. Oxidative stress

3.4.1. Intracellular ROS generationThe production of intracellular ROS was measured by DCFH

oxidation. The CM-H2DCFDA reagent passively diffuses intocells wherein it is hydrolyzed by intracellular esterase to lib-erate 2′-7′-dichlorofluoressein, which, during the reaction withoxidizing species, yields a highly fluorescent compound 2′-7′-dichlorofluorescein (DCF) that is trapped inside the cell (Liu etal., 2001). Fig. 3A clearly depicts that in comparison to controls,ROS generation increases considerably during OHBI. Mean percentDCF florescence in occult and chronic HBV was 59.65 ± 6.23% and78.62 ± 9.18%, respectively, while in controls the mean florescence

level was 0.18 ± 0.03%.

3.4.2. Depleted GR activityGlutathione reductase plays an essential role in maintaining

the appropriate levels of intracellular reduced glutathione (GSH).

A. Bhargava et al. / Virus Research 153 (2010) 143–150 147

Fig. 2. (A) DNA laddering profile showing increased apoptosis in peripheral blood lymphocytes of occult HBV and chronic HBV patients as compared with age and gendermatched healthy controls. Lanes 1 and 2: MW marker; Lane 3: control; Lanes 4 and 5: chronic HBV; Lanes 6 and 7: occult HBV. (B) Flow cytometric analysis for the mitochondrialmembrane depolarization in peripheral blood lymphocytes of healthy controls vs. occult and chronic HBV patients. (a) FSC/SSC plot showing the population of lymphocytes;(b) control cells; (c) occult HBV; (d) chronic HBV. Percent mitochondrial depolarization in cells those are positive for FL1-H (R3 zone) within a population of cells.

148 A. Bhargava et al. / Virus Research 153 (2010) 143–150

Fig. 3. (A) Flow cytometric evaluation for the induction of ROS in peripheral blood lymphocytes of controls, occult HBV and chronic HBV patients labeled with CM-H2DCFDA.( HBV;o aphica atient

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a) FSC/SSC plot showing the population of lymphocytes; (b) control cells; (c) occultf ROS within a population of cells is indicated by the shift of peak to M2 zone. (B) Grctivity in peripheral blood lymphocytes of controls, occult HBV and chronic HBV p

SH, long known for its protective function against oxidativeell damage (Collins et al., 1997), is thought to play a regula-ory role in various lymphocyte functions. Our study showedhat infection with occult HBV attributes to decline in the

ntioxidant defense mechanism leading to oxidative stress. Thebserved mean GR activity in controls was 8.73 ± 0.95 mU/mL,hile in occult and chronic HBV infected patients GR activ-

ty was 6.78 ± 1.4 mU/mL and 4.18 ± 0.72 mU/mL, respectivelyFig. 3B).

(d) chronic HBV. Cells in M1 zone are the control cells while the percent inductional representation of antioxidant defense mechanism enzyme glutathione reductases.

4. Discussion

Occult HBV is often associated with severe liver damage, lowresponse to interferon treatment and increased risk of developing

HCC (Sagnelli et al., 2008). Although, occult infections are beingreported in various clinical settings in recent times, these infec-tions still remain to be poorly characterized and their possible effecton the genomic integrity of host immune cells is unclear. Previ-ous studies had reported that chronic HBV induces genotoxic and

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utational effects on host cells via expression of certain specificroteins (Brown and Baltimore, 2003; Bakkenist and Kastan, 2004).iven that the typical HBV infections often have periodic flairs iniral replication, we aimed to explore the molecular mechanismsesulting from the genotoxic effects of OHBI on host peripherallood lymphocytes. Results of our investigation showed that infec-ion with occult HBV leads to a significant increase in the levels ofNA damage, ROS and apoptosis in peripheral blood lymphocytes.

The cellular response to DNA damage initiates a complex sig-aling cascade, which plays an important role in maintaining theenomic integrity, thereby, preventing the process of carcinogen-sis (Uziel et al., 2003; Sancar et al., 2004). Members of PI (3)-likeinase family, ATM and ATR are believed to be actively involved inommencement of the downstream signaling pathways (Ljungmannd Lane, 2004; Derheimer et al., 2007). It is supposed that insteadf directly sensing the DNA lesions they monitor secondary effectsuch as perturbation of chromatin structure or the interference ofNA metabolic processes (Ozkal et al., 2005; Lilley et al., 2007;olukbas et al., 2006; Stracker et al., 2002). Interactions of virusith the components of the complex DNA damage response net-ork possibly reveal certain proteins that regulate and determinehich pathway has to be exploited or inactivated (Kudoh et al.,

005). Recent studies have reported that viruses evolve multipleechanisms to manipulate this signaling cascade for their replica-

ion and propagation (Bartek and Lukas, 2001; Gaspar and Shenk,006; Liang et al., 2006; Zhao et al., 2008a). In addition, an in vitrotudy, performed in human hepatocyte cell line HL7702 demon-trated that HBV activates ATR checkpoint pathway without anyerceptible increase in ATM phosphorylation at Ser-1981 upon

nfection (Zhao et al., 2008b). Our study observed a considerablencrease in steady state phosphorylation of ATR and ATM in OHBIatients as compared to age and gender matched healthy controlsFig. 1C).

PI (3)-like kinases (ATM and ATR) after activation in responseo damage phosphorylates a number of downstream proteinsncluding H2AX and p53. Phosphorylation of H2AX at serine 139�H2AX) takes place immediately after the generation of a DNAreak and mediates the formation of clusters of proteins at theite of damage, known as DNA damage response foci (Kuo andang, 2008). The present study demonstrated a significant amountf �H2AX foci (serine 139) formation in peripheral lymphocytesf OHBI. Quantitative analysis revealed that H2AX phosphoryla-ion further increased in chronic HBV patients as compared withontrols (Fig. 1A and B). Besides, increased phosphorylation of p-53 was also observed in OHBI (Fig. 1C). Upon phosphorylation,53 gets stabilized and induces cell cycle arrest and/or apoptosis,ene by protecting the cells from deleterious effects of genotoxicgents (Giaccia and Kastan, 1998). In general, during apoptosis,ffector caspases are finally activated that cleave and degradeell structures, resulting in the release of apoptotic products intohe circulation. It is also evident from recent experimental datahat increased levels of circulating nucleosomes in various dis-ased states including primary biliary cirrhosis are associated withnhanced programmed cell death (Kremer et al., 2009). IncreasedNA fragmentation, along with higher levels of circulating nucle-somes observed in occult HBV patients of our study, exemplifiedhat these infections induces immunotoxic effects, which furthereads to cellular demise and might arouse a immune compromisedtate (Fig. 2A).

Moreover, viral hepatitis is often characterized by diffusednflammatory reaction, resulting in the generation of ROS (Lau et

l., 1998; Severi et al., 2007). Studies in hepatic cells have shownhat chronic HBV modulates mitochondrial function by disturbing

itochondrial membrane potential (Kim et al., 2008). Such mod-lation may prevent viral clearance or promote dissemination atifferent stages of the viral life cycle. Modulation of mitochon-

rch 153 (2010) 143–150 149

drial membrane potential is also associated with the activationof the Ca2+ uniporter which further increases ROS generation andinfluences redox-dependent signaling. This immoderate genera-tion of ROS may further cause harmful effects to biomolecules andcan directly prompt and/or adjust apoptosis (Canakci et al., 2005).Although, some studies had shown increased oxidative stress inhepatocytes during chronic HBV infections, whether a similar phe-nomenon is observed in peripheral lymphocytes during occultstate (viral load ≤100 IU/mL) remains to be elucidated (Waris andSiddiqui, 2003; Pawlak et al., 2004; Wang et al., 2004; Severi etal., 2006). The present study reported that infection with occultHBV considerably increases mitochondrial membrane depolariza-tion. The mean percentage of cells with depolarized mitochondriain occult HBV patients were 48.18 ± 4.65, while in controls andchronic HBV the levels were 0.81 ± 0.12 and 78.34 ± 8.51, respec-tively (Fig. 2B). In addition, a significant increase in the levels of ROSalong with a depleted activity of antioxidant defense enzyme GRin occult HBV patients, in comparison to their respective controls,was also reported. DCF fluorescence in occult and chronic HBV was59.65 ± 6.23 and 78.62 ± 9.18, respectively, while in controls themean fluorescence level was 0.18 ± 0.03 (Fig. 3A). While, observedlevels of GR activity in OHBI group was 6.78 ± 1.4, 4.18 ± 0.72 inchronic 8.73 ± 0.95 in control group (Fig. 3B). The results hereinclearly suggest that OHBI are strongly associated with oxidativestress, which might be the cause of increased DNA lesions andcellular demise of peripheral lymphocytes.

In conclusion, our work documents that occult HBV in spite ofits low viral load (≤100 IU/mL) induces DNA damage, apoptosis andoxidative stress in peripheral blood lymphocytes which might leadan individual to an immune compromised state. Besides provid-ing basic knowledge about the immunotoxic effects of OHBI, ourobservations might be helpful in improving the current knowledgeregarding this phenomenon. In addition, such studies may possiblyopen up a new way of identifying host cellular proteins as noveltargets to develop appropriate therapeutic strategies against viralhepatitis.

Acknowledgements

The authors are thankful to Bhopal Memorial Hospital Trust forfinancial support and thankfully acknowledge Mr. Naveen KumarKhare for providing necessary technical assistance.

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